Introduction

The
past two decades has seen remarkable advances in understanding the genetics and
pathophysiology of spontaneous animal models of immune mediated diabetes (Type
1A) including structural characterization of class II MHC presentation of insulin
peptide B:9-231 as well as the chromagranin peptide WE-14 (previously elusive target of
theBDC2.5 T cell receptor2), the creation of new animal models 3 with either genetic manipulation of autoantigens4, 5 orimmunologic function (e.g.,
toll receptor activation; peptide immunization, mutations of regulatory
pathways such as AIRE6 or foxP37), or a combination of genetic and environmental manipulation 8-18. “Humanized” mouse strains are being created and studied and promise
additional insights relevant to type 1A diabetes 19-21.In addition at the Jackson laboratory a
repository of type 1 diabetes related mouse models has been created which is an
important resource for the long-term preservation of the multiple strains that
have been created and a means to facilitate sharing of strains by different
research groups.

Spontaneous
type 1 diabetes-susceptible models include the non-obese diabetic (NOD) mouse,
the BioBreeding Diabetes-Prone (BB-DP) rat, the Komeda Diabetes-Prone (KDP)
sub-line of the Long-Evans Tokushima Lean rat Lew.1.WR1 and the Lew.1AR1/Ztm
rat. Multiple experimentally-induced models of type 1 diabetes are available
including: 1) T cell receptor (TCR) transgenic (Tg) and retrogenic mice with
the T cell receptors of naturally occurring diabetogenic clones 2) Neo-antigen
(Ag) expression under the control of the rat insulin promoter (RIP) to
establish neo-self antigen pancreatic expression that can be the target of
autoimmunity, and 3) RIP-driven expression of costimulatory molecules on beta
cells. Mice with knockouts of putative
islet autoantigens have allowed direct testing of the pathogenic significance
of specific target molecules. Strains of mice with mutations of genes
associated with type 1 diabetes in man (FoxP3 and AIRE) are being studied (including
an autosomal dominant “human” AIRE mutation6).Such strains usually do not
develop diabetes, but rather have novel autoimmune phenotypes 22-24.

A major conclusion
from these models is that type 1 diabetes is not the result of a single pathway
but can be the result of numerous distinct mechanisms 25. Nonetheless, some generalities do exist including the polygenic nature
of the disease, the involvement of T cells in the destruction of pancreatic
beta cells, and the incomplete penetrance of the disease implying that
environmental or “stochastic” factors influence disease susceptibility. These
models provide useful tools for studying the autoimmune process as well as testing
potential treatments for the prevention of type 1 diabetes. There has been some
pessimism26, appropriately expressed 10, that therapies effective in animal models such as the NOD mouse may
not be directly relevant to man. No doubt the usual animal models are inbred
(thus not diallelic at any locus) and finely “balanced” in terms of progression
to diabetes while raised in pathogen-free environments. Thus, it is likely that
many more therapies will be effective in the animal models compared to man (Entelos
has developed a computer model of NOD mice and catalogued therapeutics27). Nevertheless, the initial
experimental success of anti-CD3 monoclonal antibody therapy in man to delay
loss of insulin secretion is a direct result of studies in the NOD mouse 28, and large trials such as the Diabetes Prevention Trial (DPT) have
provided preliminary data that oral insulin (also first studied in the NOD
mouse) in subjects with elevated insulin autoantibodies may delay disease
progression to diabetes29. We suspect that informed optimism relative to the utility of animal
models is in order26 , and that the more robust the therapy in the animal model (e.g.,
ability to abrogate insulitis, ability to prevent diabetes in models engineered
to be more highly penetrant [e.g., insulin 2 gene knockout NOD mice 30) , reversal of hyperglycemia, and the closer the human studies mimic
the animal studies (e.g., dose and timing), the more relevant the study will be
to human type 1 diabetes.

The Nonobese Diabetic Mouse

There are now more than 6,000 articles listed in PubMed
on the NOD mouse making any comprehensive review of the model a difficult task
with the certainty of failure to cite all important contributions and a
certainty that not all the different pathways being pursued will be
covered.Given the ability to
electronically access primary articles through the internet with services such
as PubMed and Google my purpose in this review on such a large topic is to
provide an overall organization, a particular viewpoint (with caveats), and to attempt
to highlight many important “relevant” observations.The general hypothesis my laboratory group is
pursuing is that NOD mice 1. have relatively mild defects of immune tolerance
(particularly in comparison to foxP3 or AIRE mutant mice) and thus the
polygenic nature of the disease and the small influence of most loci; 2. that
the disease results because of an immune response that once directed at a
specific beta cell peptide is essentially normal, though the targeting leads to
disease and there is spreading of autoimmunity, with implication that all
mechanisms available to the normal immune system for destruction are operative;
3. that there is a primary autoantigenic epitope presented by the class II I-Ag7
molecule and for the NOD mouse it is the insulin B:9-23 peptide presented in
“register 3” of I-Ag731 and recognized primarily by a conserved alpha chain (TRAV5D-4) of the T
cell receptor32 and 4. The killing of islet beta cells is asynchronous with different
islets destroyed over time until enough destruction has occurred for
hyperglycemia to develop. These “biases”
are stated, as to some extent they influence “commissions” and “omissions” of
this review that many investigators will hopefully correct in the future.

The
NOD mouse is the most-studied animal model of type 1A diabetes33-37;26, 27, 38-51. The NOD inbred mouse line was established at the Shionogi Research
Laboratories in Japan through sib breeding of a hyperglycemic female mouse from
the CTS sub line 52 (Figure 1). The line that actually became the
NOD mouse strain was originally a control line for the modestly hyperglycemic
line that became the NON strain (Non-obese Non-diabetic). NOD mice show islet
infiltration by lymphocytes around 5-7 weeks of age followed by the spontaneous
development of overt diabetes in approximately 70% of the females and 40% of
the males by 30 weeks of age 53, 54. Similar to humans, NOD mice usually (but not always) express
anti-insulin autoantibodies in their serum prior to hyperglycemia 55, 56;57 (Figure 2).Of note NOR mice which do not usually develop
diabetes follow the same age dependent pattern of expression of insulin
autoantibodies.NOR mice are a complex
congenic inbred strain with approximately 80% NOD genome and following
depletion of CD4+CD25+ T cells NOR splenocytes can mediate diabetes in NOD-scid
mice58.

Figure 2. Insulin
autoantibodies and glucose of individual NOD mice followed from 4 weeks of age.

Although
all NOD mice develop insulitis, this is not always followed by diabetes.
Diabetes incidence varies among established NOD colonies, although in all
cases, unlike in humans, gender influences disease incidence with higher
disease frequencies in female mice than males 54. Type 1 diabetes incidence is also affected by
environmental conditions with higher disease frequencies in sterile conditions 59 and lower disease penetrance with infectious agents 60, 61. The disease can be prevented in numerous ways (>134) 26 including immunological or genetic manipulations of NOD mice 62.

Diabetes
susceptibility in the NOD mouse results from the interaction of multiple genes63, with the strongest predisposing effect deriving from genes within the
major histocompatibility complex (MHC) 64-66. Multiple studies suggest that the effect of the MHC 64 is due to the combination of the unique sequence of
the class II I-Ag7 allele with its beta chain lacking aspartic acid
at position 57 and proline at position 56 67 (the I-Aalpha chain sequence of NOD is identical to
BALB/c), a lack of expression of I-E 68 due to a common mouse mutation (also present in
C57BL/6 mice) and specific class I alleles 69, with additional polymorphisms of other genes within the MHC(e.g. idd1670, 71. In general it has been assumed
that the lack of aspartic acid at position 57 of the I-Ag7 beta
chain creating a basic I-Ag7 pocket 9 would favor the binding of
amino acids with negatively charged side chains into pocket 9. The recognition of these peptides by the CD4 T
cells then creates class II MHC mediated diabetes susceptibility.Kappler and coworkers have recently
discovered that for the insulin peptide B:9-23, just the opposite occurs.A given peptide can bind to I-Ag7
in different registers with side chains of different amino acids of the peptide
binding in pockets 1, 4, 6 and 9 depending on where the peptide docks along the
linear I-Ag7 groove (register).T cell receptors then recognize the peptide MHC complex in a specific
register. For each specific register
that a single peptide binds in, the amino acid chains facing the T cell
receptor are different.To go from one
register to another, the peptide must rotate for specific side chains to bind
in pockets 1,4,6 and 9.By covalently coupling the B:9-23 peptide in
the groove of I-Ag7 with the arginine (peptide amino acid 22) in
pocket 9 (unfavored basic amino acid in a basic pocket) it was found that all
of the studied anti-B:9-23 autoreactive T cells reacted only when the peptide
was in this low affinity register1.This has led to the hypothesis
that such unusual recognition may allow anti-B:9-23 T cells to avoid thymic
deletion in that the concentrations of insulin in the thymus are very low.T cells should be able to recognize the
peptide in a low affinity register in the islets where the concentrations of
insulin and presumably the B:9-23 peptide presented by I-Ag7 are
huge.Of note Unanue and coworkers have
provided data that the B:9-23 peptide is likely created within islets,
potentially within insulin secretory granules with subsequent uptake by antigen
presenting cells72.

A
great deal of effort has been involved in mapping the diabetes-associated
alleles in both NOD mice and humans. In both cases, numerous loci have been
identified (>50), although to date,few predisposing genes are identified for the NOD mouse (MHC genes 64, potentially CTLA-4 73, IL263, 74 and beta-2 microglobulin important exceptions 66, 75, 76). Genetic analysis has involved mapping of the mouse genome usually
with crossing of loci from nonautoimmune-prone strains onto the NOD background
as well as analysis of recombinant and related strains. Loci (identified as Iddn in the mouse and IDDMn in the human) with significant LOD
scores for association have been analyzed for candidate genes. Multiple
congenic strains containing Idd locus (n) from disease-resistant strains have
been generated and analyzed for disease incidence, insulitis, andimmunological phenotypes (e.g., insulin
autoantibodies 77) associated with diabetes in the NOD mouse 76,
78, 79. A general finding is that several identified regions
contain multiple-disease associated loci which alone rarely confer protection
but in multitudes of two or more can nearly completely block disease 76. Positional cloning of numerous Idd loci is underway with potential for
success of identifying “causative” polymorphisms likely to be dependent upon
the number of genes within a given locus. For example a 1.52Mb region of locus
Idd5.2 contains 45 genes, while the locus Idd5.1 (2.1-Mb region) contains four
genes, including CTLA-4 63, with evidence for influence of CTLA-4 polymorphisms influencing
disease in man and mouse.

A
list of many of the multiple identified Idd loci, their positions on mouse
chromosomes, identified phenotypes, and candidate genes is presented in Table
3.1 and a more comprehensive list is provided by Serreze and coworkers 37. These loci can either confer susceptibility or
resistance to disease development and even some alleles of the NOD mouse confer
resistance rather than susceptibility. Given the identification of these loci,
it is possible to rapidly introduce loci from other strains onto the NOD
background to create what has been termed speed congenics. Usually within five
generations of backcrossing, essentially all NOD diabetogenic loci can be fixed
with the locus of interest introduced 80. Though there are reports of some success, a major
disadvantage of the NOD mouse is the lack of embryonic stem cell lines to
directly create gene knockouts on the NOD background, a lack that is partially
alleviated with the availability of “mixed” NOD strain embryonic cell lines 81.